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  • 1.
    Akhunzianov, Almaz A.
    et al.
    Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation.
    Nesterova, Alfiya I.
    Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation; Republican Clinical Oncology Dispensary Named after Prof. M.Z. Sigal, Kazan, Russian Federation.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Filina, Yulia V.
    Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation.
    Rizvanov, Albert A.
    Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation.
    Miftakhova, Regina R.
    Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation.
    Unravelling the Therapeutic Potential of Antibiotics in Hypoxia in a Breast Cancer MCF-7 Cell Line Model2023In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 14, article id 11540Article in journal (Refereed)
    Abstract [en]

    Antibiotics inhibit breast cancer stem cells (CSCs) by suppressing mitochondrial biogenesis. However, the effectiveness of antibiotics in clinical settings is inconsistent. This inconsistency raises the question of whether the tumor microenvironment, particularly hypoxia, plays a role in the response to antibiotics. Therefore, the goal of this study was to evaluate the effectiveness of five commonly used antibiotics for inhibiting CSCs under hypoxia using an MCF-7 cell line model. We assessed the number of CSCs through the mammosphere formation assay and aldehyde dehydrogenase (ALDH)-bright cell count. Additionally, we examined the impact of antibiotics on the mitochondrial stress response and membrane potential. Furthermore, we analyzed the levels of proteins associated with therapeutic resistance. There was no significant difference in the number of CSCs between cells cultured under normoxic and hypoxic conditions. However, hypoxia did affect the rate of CSC inhibition by antibiotics. Specifically, azithromycin was unable to inhibit sphere formation in hypoxia. Erythromycin and doxycycline did not reduce the ratio of ALDH-bright cells, despite decreasing the number of mammospheres. Furthermore, treatment with chloramphenicol, doxycycline, and tetracycline led to the overexpression of the breast cancer resistance protein. Our findings suggest that hypoxia may weaken the inhibitory effects of antibiotics on the breast cancer model.

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  • 2. Al-Behadili, Ali
    et al.
    Uhler, Jay P.
    Berglund, Anna-Karin
    Peter, Bradley
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reyes, Aurelio
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zeviani, Massimo
    Falkenberg, Maria
    A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 18, p. 9471-9483Article in journal (Refereed)
    Abstract [en]

    The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.

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  • 3.
    Al-Furoukh, Natalie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ianni, Alessandro
    Nolte, Hendrik
    Hölper, Soraya
    Krüger, Marcus
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Braun, Thomas
    ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells2015In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1853, no 10, p. 2580-2591Article in journal (Refereed)
    Abstract [en]

    Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondria( proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.

  • 4.
    Andréasson, Måns
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Donzel, Maxime
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Abrahamsson, Alva
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Berner, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Clinical Genetics Unit, Department of Women and Children’s Health, Padua University, Padua, Italy.
    Quiroga, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Eriksson, Anna U.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chao, Yu-Kai
    Mechanistic and Structural Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, United Kingdom.
    Overman, Jeroen
    Mechanistic and Structural Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, United Kingdom.
    Pemberton, Nils
    Medicinal Chemistry, Research and Early Development, Respiratory and Immunology (R&I), Bio Pharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Exploring the dispersion and electrostatic components in arene-arene interactions between ligands and G4 DNA to develop G4-ligands2024In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 67, no 3, p. 2202-2219Article in journal (Refereed)
    Abstract [en]

    G-Quadruplex (G4) DNA structures are important regulatory elements in central biological processes. Small molecules that selectively bind and stabilize G4 structures have therapeutic potential, and there are currently >1000 known G4 ligands. Despite this, only two G4 ligands ever made it to clinical trials. In this work, we synthesized several heterocyclic G4 ligands and studied their interactions with G4s (e.g., G4s from the c-MYC, c-KIT, and BCL-2 promoters) using biochemical assays. We further studied the effect of selected compounds on cell viability, the effect on the number of G4s in cells, and their pharmacokinetic properties. This identified potent G4 ligands with suitable properties and further revealed that the dispersion component in arene-arene interactions in combination with electron-deficient electrostatics is central for the ligand to bind with the G4 efficiently. The presented design strategy can be applied in the further development of G4-ligands with suitable properties to explore G4s as therapeutic targets.

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  • 5.
    Andréasson, Måns
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Donzel, Maxime
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Abrahamsson, Alva
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Berner, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quiroga, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pemberton, Nils
    AstraZeneca, Gothenburg, Sweden.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The Synergism of the Dispersion and Electrostatic Components in the Arene-Arene Interactions Between Ligands and G4 DNAManuscript (preprint) (Other academic)
  • 6.
    Berner, Andreas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bhuma, Naresh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Golebiewska, Justyna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Abrahamsson, Alva
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andréasson, Måns
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chaudhari, Namrata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bose, Partha Pratim
    Department of Biosciences and Nutrition, Karolinska Institutet, Neo, Huddinge, Sweden.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Strömberg, Roger
    Department of Biosciences and Nutrition, Karolinska Institutet, Neo, Huddinge, Sweden.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    G4-ligand-conjugated oligonucleotides mediate selective binding and stabilization of individual G4 DNA structures2023In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 146, no 10, p. 6926-6935Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

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  • 7. Boldinova, Elizaveta O.
    et al.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Khairullin, Rafil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Kazan Volga Reg Fed Univ, Russia.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol2017In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 9, article id e0184489Article in journal (Refereed)
    Abstract [en]

    Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37 degrees C. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

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  • 8. Boldinova, Elizaveta O.
    et al.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shilkin, Evgeniy S.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol2017In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 18, no 7, article id 1584Article, review/survey (Refereed)
    Abstract [en]

    PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

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  • 9.
    Calvo, Patricia A
    et al.
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Martínez-Jiménez, María I
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Díaz, Marcos
    Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kasho, Kazutoshi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain; Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
    Guerra, Susana
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Méndez, Juan
    Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
    Blanco, Luis
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart2021In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no 14, p. 8199-8213Article in journal (Refereed)
    Abstract [en]

    PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

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  • 10.
    Das, Rabindra Nath
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Berner, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bhuma, Naresh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Golebiewska, Justyna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Abrahamsson, Alva
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andréasson, Måns
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chaudhari, Namrata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Development of a G4 Ligand-Conjugated Oligonucleotide Modality that Selectively Targets Individual G4 DNA StructuresManuscript (preprint) (Other academic)
  • 11.
    Doimo, Mara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Women and Children Health, University of Padova, Padova, Italy.
    Chaudhari, Namrata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Abrahamsson, Sanna
    Bioinformatics and Data Centre, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    L'Hôte, Valentin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nguyen, Tran V. H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berner, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ndi, Mama
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Abrahamsson, Alva
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Aasumets, Koit
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    Goffart, Steffi
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    Pohjoismäki, Jaakko L. O.
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    López, Marcela Dávila
    Bioinformatics and Data Centre, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 14, p. 7392-7408Article in journal (Refereed)
    Abstract [en]

    Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

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  • 12.
    Doimo, Mara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University.
    MtDNA replication, maintenance, and nucleoid organization2020In: The human mitochondrial genome: from basic biology to disease / [ed] Giuseppe Gasparre; Anna Maria Porcelli, Academic Press, 2020, p. 3-33Chapter in book (Refereed)
    Abstract [en]

    Part of the genetic information in human cells resides in the mitochondria. Faithful maintenance of mitochondrial deoxyribonucleic acid (mtDNA) is crucial for the oxidative phosphorylation system that produces the majority of the cellular ATP, and therefore to life. This chapter provides an introduction into the characteristics of human mtDNA and summarizes the processes and factors required for the replication and maintenance of this small but essential genome. We also describe the organization of mtDNA in specialized nucleoprotein structures called nucleoids. Where applicable, we refer to human disease states that are caused by defects in the described factors or processes.

  • 13.
    Forslund, Josefin M. E.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovič, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Pauline H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The presence of rNTPs decreases the speed of mitochondrial DNA replication2018In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 3, article id e1007315Article in journal (Refereed)
    Abstract [en]

    Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondnal DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol gamma) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol gamma exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol gamma and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol gamma reported here could have implications for forms of rntDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTPidNIP ratios that are expected to prevail in such disease states, Pol gamma enters a polymerasetexonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

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  • 14.
    Inatomi, Teppei
    et al.
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Matsuda, Shigeru
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan; Department of Modomics Biology and Medicine, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryocho, Aoba-ku, Miyagi, Sendai-shi, Japan.
    Ishiuchi, Takashi
    Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Do, Yura
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Nakayama, Masunari
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Abe, Shusaku
    Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Kasho, Kazutoshi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nakada, Kazuto
    Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Ibaraki, Tsukuba-shi, Japan.
    Ichiyanagi, Kenji
    Department of Animal Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Aichi, Nagoya-shi, Japan.
    Sasaki, Hiroyuki
    Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    Yasukawa, Takehiro
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan; Department of Pathology and Oncology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan.
    Kang, Dongchon
    Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka-shi, Japan.
    TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication2022In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1869, no 1, article id 119167Article in journal (Refereed)
    Abstract [en]

    Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

  • 15.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Rajendra
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Akhunzianov, Almaz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia.
    Deiana, Marco
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kasho, Kazutoshi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sulis Sato, Sebastian
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pourbozorgi-Langroudi, Parham
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mason, James E.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Medini, Paolo
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Öhlund, Daniel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization2020In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, no 6, p. 2876-2888Article in journal (Refereed)
    Abstract [en]

    The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

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  • 16.
    Kasho, Kazutoshi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Krasauskas, Lukas
    Life Sciences Center, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
    Smirnovas, Vytautas
    Life Sciences Center, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Human polymerase δ-interacting protein 2 (Poldip2) inhibits the formation of human tau oligomers and fibrils2021In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, no 11, article id 5768Article in journal (Refereed)
    Abstract [en]

    A central characteristic of Alzheimer’s disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.

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  • 17.
    Kasho, Kazutoshi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Velázquez-Ruiz, Cristina
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Martínez-Jiménez, Maria Isabel
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Laurent, Timothée
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berner, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pérez-Rivera, Aldo E.
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Jenninger, Louise
    Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
    Blanco, Luis
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol2021In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no 4, p. 2179-2191Article in journal (Refereed)
    Abstract [en]

    Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.

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  • 18. Miralles Fusté, Javier
    et al.
    Shi, Yonghong
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhu, Xuefeng
    Jemt, Elisabeth
    Persson, Orjan
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gustafsson, Claes M
    Falkenberg, Maria
    In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, p. e1004832-Article in journal (Refereed)
    Abstract [en]

    Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.

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  • 19.
    Moodie, Lindon W. K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Xin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Albers, Michael Franz
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Photoactivated Colibactin Probes Induce Cellular DNA Damage2019In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 5, p. 1417-1421Article in journal (Refereed)
    Abstract [en]

    Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks(+) bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative "warhead" and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks(+) like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

  • 20. Pohjoismäki, Jaakko L. O.
    et al.
    Forslund, Josefin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Goffart, Steffi
    Torregrosa-Muñumer, Rubén
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Known Unknowns of Mammalian Mitochondrial DNA Maintenance2018In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 40, no 9, article id 1800102Article in journal (Refereed)
    Abstract [en]

    Mammalian mitochondrial DNA (mtDNA) replication and repair have been studied intensively for the last 50 years. Although recently advances in elucidating the molecular mechanisms of mtDNA maintenance and the proteins involved in these have been made, there are disturbing gaps between the existing theoretical models and experimental observations. Conflicting data and hypotheses exist about the role of RNA and ribonucleotides in mtDNA replication, but also about the priming of replication and the formation of pathological rearrangements. In the presented review, we have attempted to match these loose ends and draft consensus where it can be found, while identifying outstanding issues for future research.

  • 21.
    Prasad, Bagineni
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andréasson, Måns
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    L'Hôte, Valentin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A complementary chemical probe approach towards customized studies of G-quadruplex DNA structures in live cells2022In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 13, no 8, p. 2347-2354Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform to generate complementary chemical probes centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design and substantial synthetic devlopments to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. We next used this reactive handle to conjugate a BODIPY fluorophore to Phen-DC3. This generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effect that revealed the localization and function of Phen-DC3 in human cells. To increase cellular uptake, a second chemical probe with a conjugated cell-penetrating peptide was prepared using the same amine-substituted Phen-DC3 derivative. The cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this method to modulate and direct cellular uptake e.g. as delivery vehicles. The applied approach to generate multiple tailored biochemical tools based on the same core structure can thus be used to advance the studies of G4 biology to uncover molecular details and therapeutic approaches. This journal is

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  • 22.
    Stojkovic, Gorazd
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
    Forslund, Josefin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Burgers, Peter M.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
    Oxidative DNA damage stalls the human mitochondrial replisome2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 28942Article in journal (Refereed)
    Abstract [en]

    Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase. holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.

  • 23. Torregrosa-Muñumer, Rubén
    et al.
    Forslund, Josefin M. E.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Goffart, Steffi
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovič, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carvalho, Gustavo
    Al-Furoukh, Natalie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blanco, Luis
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pohjoismäki, Jaakko L. O.
    PrimPol is required for replication reinitiation after mtDNA damage2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 43, p. 11398-11403Article in journal (Refereed)
    Abstract [en]

    Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

  • 24.
    Wanrooij, Paulina H.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Engqvist, Martin K. M.
    Forslund, Josefin M. E.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Navarrete, Clara
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sedman, Juhan
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clausen, Anders R.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 47, p. 12466-12471, article id 201713085Article in journal (Refereed)
    Abstract [en]

    Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

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